Biochip with a three dimensional mesoporous layer and method for forming the same

ABSTRACT

The present invention discloses a biochip comprising a substrate and a three-dimensional mesoporous layer on top of it, wherein the mesoporous layer on top is chemically modified to recognize labeling DNAs, proteins, peptides, saccharides, and cells. In addition, this invention also discloses a method for forming the biochip with a three-dimensional mesoporous layer, including a blending process, a heating process, a coating process, a gelation process, a drying process, and a surface modification process.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is generally related to a biochip, and moreparticularly to a biochip with a three-dimensional mesoporous layer anda method for forming the same.

2. Description of the Prior Art

At present, the biochip detection technology becomes increasinglyimportant in biotechnology. The biochip detection technology cansimultaneously detect various pathogens on a single chip and break thedetection limitation achieved by traditional technologies. Amicroarrayed biochip is generally prepared by aligning a large quantityof bio-probes (DNA's or proteins) on a chip substrate and is used foranalyzing or testing samples by the hybridization of DNA-DNA or specificbinding between proteins. According to the detection objectives, thereare two major categories for microarrayed biochips: DNA chip and proteinchip. DNA chips use nucleotide molecules as the probes to detect theirnucleotide fragments. DNA chips can also be categorized intocomplimentary DNA (cDNA) chips and oligonucleotide chips, according tothe length of the probes spotted on chips. cDNA chips are often used inthe research of gene expressions; while oligonucleotide chips can alsobe used in diagnosis of pathogen and genotyping in addition to geneexpression analysis.

For DNA chips, probes are immobilized on substrates and used to detectspecific DNA fragments by the characteristic hybridization withcomplimentary DNA's. DNA chips can be applied on disease detection andshorten the time for developing new medicines. DNA chip is also apowerful tool for analyzing DNA's by appropriate dye labeling in visibleemission lights. By different emission wavelengths, individual targetDNA can be distinguished and analyzed.

The improvement of detection sensitivity by modifying the substratesurfaces of traditional biochips is currently still being sought toobtain amplified signals to facilitate further analysis. Thus, a novelbiochip preparation method is proposed to achieve the high-sensitivityperformance.

SUMMARY OF THE INVENTION

In accordance with the present invention, a biochip with athree-dimensional mesoporous layer and a method for forming the same areprovided.

The three-dimensional mesoporous material is a network polymer withnano-scaled pores, such as aerogel material. Its porosity can be as highas 95%. Due to its high porosity, it possesses a variety ofcharacteristics: high specific surface area, low density, low heatconductivity, low sound spreading speed, low dielectric constant, and soforth. Therefore, it can be applied in various fields, such as heatinsulation, catalyst, adsorbent, electrodes, electronics, detectors,etc.

The first objective of the present invention is to synthesize materialson the top of a flat substrate to form a three-dimensional mesoporouslayer using the sol-gel technique.

The second objective of the present invention is to utilize the largethree-dimensional inner specific surface area to recognize labeled DNAs,proteins, peptides, saccharides, and cells. Thus, the biochip with athree-dimensional mesoporous layer according to the present inventionhas the characteristics of high sensitivity of detection so as it wouldhave a potential to simplify the detection equipments. For example, onlydata type camera (CCD) would be required instead of complicated imagingtechnique. Therefore, this present invention does have the economicpotential for industrial applications.

Accordingly, the present invention discloses a biochip comprising asubstrate and a three-dimensional mesoporous layer on top of thesubstrate. The surface of the three-dimensional mesoporous layer ischemically modified to recognize labeled DNAs, proteins, peptides,saccharides, and cells. In addition, this invention also discloses amethod for preparing the biochip with a three-dimensional mesoporouslayer, including a blending process, a heating process, a coatingprocess, a gelation process, a cleaning process, a drying process, and asurface modification process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a picture showing the slide with a three-dimensional aerogellayer according to a preferred example of the present invention;

FIG. 2 shows the measurement data of nitrogen adsorption/desorption ofthe three-dimensional aerogel layer before the modification according toa preferred example of the present invention;

FIG. 3 shows the measurement data of nitrogen adsorption/desorption ofthe three-dimensional aerogel layer after modified by GLYMO according toa preferred example of the present invention;

FIG. 4 shows the TGA comparison data of the three-dimensional aerogellayer before/after modification according to a preferred example of thepresent invention;

FIG. 5 shows SEM (scanning electron microscope) images of the surface ofthe three-dimensional aerogel layer according to a preferred example ofthe present invention;

FIG. 6 shows the analysis result of the unmodified three-dimensionalaerogel layer according to a preferred example of the present inventionby the ²⁹Si solid-state nuclear magnetic resonance spectrometer;

FIG. 7 is a scanned image of the three-dimensional aerogel layermodified by 10% GLYMO according to a preferred example of the presentinvention; and

FIG. 8 is a scanned image of the three-dimensional aerogel layermodified by 10% GLYMO and then dripped with Cy5-labeled Bac-alP1 oligoDNA according to a preferred example of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

What is probed into the invention is a biochip with a three-dimensionalmesoporous layer and a method for forming the same. Detail descriptionsof the structure and elements will be provided as followed in order tomake the invention thoroughly understood. The application of theinvention is not confined to specific details familiar to those who areskilled in the art. On the other hand, the common structures andelements that are known to everyone are not described in details toavoid unnecessary limits of the invention. Some preferred embodiments ofthe present invention will now be described in greater detail asfollowed. However, it should be recognized that the present inventioncan be practiced in a wide range of other embodiments besides thoseexplicitly described, that is, this invention can also be appliedextensively to other embodiments, and the scope of the present inventionis expressly not limited except as specified in the accompanying claims.

In one embodiment of the present invention, a method for forming abiochip with a three-dimensional mesoporous layer is disclosed. Atfirst, a precursor solution is provided. The precursor solutioncomprises an ionic liquid and at least one alkoxide monomer and/oraryloxide monomer. The ionic liquid is used as a template as well as asolvent. The central element of the alkoxide monomer and/or aryloxidemonomer comprises one selected from the group consisting of thefollowing elements: Li, Na, K, Rb, Cs, Be, Mg, Ca, Sr, Ba, Ti, Te, Cr,Cu, Er, Fe, Ta, V, Zn, Zr, Al, Si, Ge, Sn, and Pb. Next, a coatingprocess is carried out to coat the precursor solution on a substrate.The material of the substrate comprises one selected from the groupconsisting of the following materials: silicon chip, glass, or polymer.The precursor solution also comprises an acidic compound or alkalinecompound to catalyze the hydrolysis/polymerization of the alkoxidemonomer and/or aryloxide monomer. The method for preparing the precursorsolution described as followed: firstly blending the alkoxide monomerand/or aryloxide monomer and the ionic liquid together to form a firstmixture; next adding an acidic compound to the first mixture to form asecond mixture; and finally adding an alkaline compound to the secondmixture to enhance the hydrolysis/polymerization reactions of thealkoxide monomer and/or aryloxide monomer.

After the coating process, a gelation process is carried out by havingan alcohol contact with the precursor solution on the substrate so as toform a gel membrane. The temperature of the gelation process ranges from50° C. to 150° C. Then, a cleaning process is carried out to remove theionic liquid from the gel membrane by a solvent. The temperature of thecleaning process ranges from 30° C. to 200° C. Following that, a dryingprocess is performed to remove the solvent to form the three-dimensionalmesoporous layer. The preferred solvent is the one with a low boilingpoint (less than or equal to 200° C.) Preferably, the solvent comprisesone selected from the group consisting of the following: nitrile,alcohol, ketone, and water. The common composition of thethree-dimensional mesoporous layer comprises one selected from the groupconsisting of the following or any combination: SiO₂, TiO₂, V₂O₅, andAl₂O₃. The average pore diameter of the three-dimensional mesoporouslayer ranges about 2 nm to 50 nm. The specific surface area is more thanor equal to 100 m²/g and the porosity is 50%˜99%. Finally, a surfacemodification process is carried for the three-dimensional mesoporouslayer of the composite membrane, so as to form a biochip with athree-dimensional mesoporous layer.

In the embodiment, the mentioned ionic liquids are room temperatureionic liquids (RTIL's), and is formed by mixing an organic base with aLewis acid. When the Lewis acid is halogenated acid, it can form a roomtemperature ionic liquid but will produce halogen acid if reacting withwater. Therefore, the halogenated acid is not suitable for the presentinvention. The Lewis acid used by the present invention is nothalogenated acid so as to prepare a stable ionic liquid in water. In apreferred example, the cationic moiety in the organic base is alkyl oraryl group having the following general equation:

in which R¹, R², R³, and R⁴ are selected according to the followingtable.

R¹ R² R³ R⁴ CH₃ H CH₃ H C₂H₅ H CH₃ H C₂H₅ H C₂H₅ H CH₃CH₂CH₂CH₂ H CH₃ H(CH₃)₂CHCH₂ H CH₃ H CH₃CH₂CH₂CH₂ H C₂H₅ H CH₃ H CH₃OCH₂CH₂ H CH₃ HCF₃CH₂ H CH₃ CH₃ C₂H₅ H CH₃ CH₃ CH₃CH₂CH₂ H C₆H₆CH₂ CH₃ CH₃CH₂CH₂ HC₆H₆CH₂ CH₃ CH₃CH₂CH₂CH₂ H C₆H₆CH₂ CH₃ (CH₃CH₂)(CH₃)CH H C₆H₆CH₂ CH₃CH₃CH₂CH₂CH₂CH₂ H CH₃ H C₂H₅ CH₃ C₂H₅ H C₂H₅ CH₃For example, the common organic cationic moiety comprises one selectedfrom the group consisting of the following:1-n-butyl-3-methylimidazolium (BMI), 1-octanyl-3-methylimidazolium(OMI), 1-dodecanyl-3-methylimidazolium (DMI), and1-hexadecanyl-3-methylimidazolium (HDMI). In addition, the anionicmoiety in the Lewis acid comprises one selected from the groupconsisting of the following: BF₄ ⁻, PF₆ ⁻, SbF₆ ⁻, F(HF)_(n) ⁻, CF₃SO₃⁻, CF₃CF₂CF₂CF₂SO₃ ⁻, (CF₃SO₂)₂N⁻[TFSI], (CF₃SO₂)₃C⁻, CF₃COO⁻, andCF₃CF₂CF₂COO⁻. When the cationic moiety to be used is determined, theanionic moiety in the Lewis acid can be adjusted to controlhydrophilicity/hydrophobicity. For example, BMI-BF₄ is hydrophilic andBMI-TFSI is hydrophobic.

For instance, alkyloxide monomer is used as an example. Alkyloxidemonomer is hydrolyzed to form hydrophilic silanol (—Si—O—H). Thus, thehydrophilic ionic liquid and silanol are tended to attract to each otherand can stabilize the formation of silicon oxide structure so as toobtain more stable three-dimensional silicon oxide mesoporous material.In this embodiment, the weight of the ionic liquid is about 10%˜70%weight of the at least alkoxide monomer and preferably about 20%˜50%.When the added amount is more than the upper limit, the solconcentration is reduced and the gelation is slow to result in unstablestructure.

In this embodiment, when the central element of the alkoxide monomerkeeps the same, the specific surface area of the three-dimensionalmesoporous material increases with the increase of the molecular weightof the non-alkoxyl block in the alkoxide monomer. For example, in thecase of alkoxide monomer, when tetramethyl orthosilicate (TMOS),tetraethyl orthosilicate (TEOS), bis(triethoxysilyl)ethane (BTSE), andbis(triethoxysilyl)benzene (BTSB) are separately used as the precursor,it is found that the specific surface area of the three-dimensionalmesoporous material by using BTSE or BTSB is greater than that by usingTMOS or TEOS. It is speculated that the increase of the quantities ofpores in the three-dimensional mesoporous material as well as theincrease of the specific surface area are because the vinyl group ofBTSE and the phenyl group of the BTSB occupy the networking space in thethree-dimensional mesoporous material.

In this embodiment, the coating process comprises a heating process toheat the precursor solution. The operating temperature of the heatingprocess ranges from 40° C. to 70° C. to form an early stage gel.Besides, the gelation process comprises a gas contacting process and aliquid contacting process. In the gas contacting process, the saturatedvapor of the alcohol is in contact with the early stage gel formed bythe precursor solution on the substrate to form a stable gel. Followingthat, in the liquid contacting process the alcohol solution is incontact with the gel layer so as to harden the stable gel layer withoutshrinkage during the gelation and form said three-dimensional mesoporouslayer. The number of carbon atoms in the alcohol is less than or equalto 5.

The modifier for the surface modification process is an alkoxide monomerand/or aryloxide monomer with at least one specific moiety. The specificmoiety comprises one selected from the group consisting of thefollowing: amine group, hydroxyl group, carboxyl group, and epoxy group.The common modifier comprisesN-[3-(trimethoxysilyl)propyl]-1,2-ethanediamine (DAMO),3-Glycidoxypropyl-trimethoxysilane (GLYMO), 3-Aminopropyltriethoxysilane(APTS), N-(2-Aminoethyl)3-aminopropyltriethoxysilane (TMsen) and soforth. According to a preferred example of the present invention, afterthe surface modification process, a converting process is carried out.At first, a converter that comprises a first moiety and a second moietyis provided. Then, the specific moiety of the modifier is bonded withthe first moiety of the converter to form a biochip having the secondmoiety on its surface. For example, when the modifier isN-[3-(trimethoxysilyl)propyl]-1,2-ethanediamine (DAMO), glutaraldehydecan be used as the converter to form the mesoporous layer havingaldehyde group on its surface.

EXAMPLE

According to a preferred example of the present invention, the methodfor forming a biochip with a three-dimensional aerogel layer isprovided. The method comprises the following steps.

(1) Cleaning a Glass Slide:

The glass slide is rinsed by sodium hydroxide, deionized water and iscleaned in a supersonic oscillation tank. It is then taken out and thendried.

(2) Synthesizing an Aerogel Layer on the Surface of the Glass Slide:

a. Formic acid, ionic liquid (used as the molecular template), andtetraethylsiloxane with a certain ratio are blended and heated to 60° C.by a hot-water bath.

b. The solution in step a is coated onto the surface of the cleanedglass slide by injection. The coated glass slide is placed in acontainer having saturated ethanol vapor pressure. Then ethanol (itstemperature is more than or equal to 60° C.) is added to cover thetopmost aerogel layer on the glass slide. It is then stood still forshaping.

c. The slide is then taken out after shaping and rinsed with a lowboiling point solvent. After one day, it is taken out and dried.

(3) Modifying the Surface of the Three-Dimensional Aerogel Layer:

N-[3-(trimethoxysilyl)propyl]-1,2-ethanediamine (DAMO) andglutaraldehyde with different concentrations are used as the first typemodifier and 3-Glycidoxypropyl-trimethoxysilane (GLYMO) is used as thesecond type modifier.

After the above steps (1)˜(3), the slide with an aerogel layer isformed. GenePix400B chip scanner is used to scan the background and thefluorescence intensity of the Cy5-labeled Bac-alP1 oligo DNA (60 mer).FIG. 1 shows the outward appearance of the glass slide with the aerogellayer. White powder-like substance is seen on the surface. If clearwater is dropped on the glass, it is observed that water diffusesquickly into the inner layer. Referring to FIG. 2, the specific surfacearea of the unmodified aerogel layer reaches 717 m²/g and the averagepore diameter is 9.4 nm. Porosity is as high as 96% and the volume ofthe pore is also as high as 2.0 cm³/g.

FIG. 3 shows the result of the aerogel layer after modified by GLYMO.Its specific surface area is 344 m²/g and the average pore diameter is6.1 nm. The volume of the pore is reduced to 0.4 cm³/g. Therefore, thesurface modification by GLYMO modifier does not completely block thepores.

Referring to FIG. 4, from the thermogravimetric experiment, the weightafter modification loses about 15%, compared to that beforemodification. It indicated that the bonded GLYMO is about 15% weight ofthe aerogel layer. FIG. 5 shows SEM (scanning electron microscope) imageof the surface of the aerogel layer.

Referring to FIG. 6, the unmodified three-dimensional aerogel layer hasa distinctive peak at −107˜110 ppm, that is the four-substituted silicacharacteristic peak of the ²⁹Si solid-state nuclear magnetic resonancespectrum. As shown in FIG. 6, the major distinctive peak appears around−111.1 ppm, that represents four-substituted Si (Q₄), and the rest peakaround −100 ppm represents three-substituted Si (Q₃), so as to formsilica network crosslinking structure.

FIG. 7 is a scanned image of the three-dimensional aerogel layermodified by 10% GLYMO. From the observed blue uniform signal, thebackground interference caused by the aerogel layer on the glass slideis very small. FIG. 8 is a scanned image of the aerogel layer modifiedby 10% GLYMO and then dripped with Cy5 labeled Bac-alP1 oligo DNA. It isfound that Cy5 fluorescein shows in light blue circular dots.

Other modifications and variations are possibly developed in light ofthe above demonstrations. It is therefore to be understood that withinthe scope of the appended claims the present invention can be practicedotherwise than as specifically described herein. Although specificembodiments have been illustrated and described herein, it is obvious tothose skilled in the art that many modifications of the presentinvention may be made without departing from what is intended to belimited solely by the appended claims.

1. A method for forming a biochip with a three-dimensional mesoporous layer, comprising: providing a precursor solution that comprises an ionic liquid and at least one alkoxide monomer and/or aryloxide monomer; performing a coating process to coat said precursor solution on a substrate to form an early stage gel; performing a gelation process to form a three-dimensional mesoporous layer by having an alcohol contact with said early stage gel formed by the said precursor solution on said substrate, so as to form a composite membrane with a three-dimensional mesoporous layer; performing a cleaning process by a solvent to remove said ionic liquid in said three-dimensional mesoporous layer from said composite membrane; performing a drying process to remove the solvent in said three-dimensional mesoporous layer from said composite membrane; and performing a surface modification process for said three-dimensional mesoporous layer, so as to form a biochip with a three-dimensional mesoporous layer.
 2. The method according to claim 1, wherein said precursor solution further comprises an acidic compound or alkaline compound.
 3. The method according to claim 1, wherein the method for forming said precursor solution comprises: blending said alkoxide monomer and/or aryloxide monomer and said ionic liquid together to form a first mixture; adding an acidic compound to said first mixture to form a second mixture; and adding an alkaline compound to said second mixture to form said precursor solution.
 4. The method according to claim 1, wherein the central element of said alkoxide monomer and/or aryloxide monomer comprises one selected from the group consisting of the following: Li, Na, K, Rb, Cs, Be, Mg, Ca, Sr, Ba, Ti, Te, Cr, Cu, Er, Fe, Ta, V, Zn, Zr, Al, Si, Ge, Sn, and Pb.
 5. The method according to claim 1, wherein said alkoxide monomer and/or aryloxide monomer comprises one selected from the group consisting of the following: tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS), bis(triethoxysilyl)ethane (BTSE), bis(triethoxysilyl)benzene (BTSB), and tetrabutyl titanate (TBOT).
 6. The method according to claim 1, wherein said ionic liquid is a room temperature ionic liquid.
 7. The method according to claim 1, wherein said ionic liquid is formed by mixing an organic base with a Lewis acid that is not halogenated acid.
 8. The method according to claim 7, wherein the cationic moiety in said organic base is alkyl or aryl group having the following general equation:

in which R¹, R², R³, and R⁴ are selected according to the following table. R¹ R² R³ R⁴ CH₃ H CH₃ H C₂H₅ H CH₃ H C₂H₅ H C₂H₅ H CH₃CH₂CH₂CH₂ H CH₃ H (CH₃)₂CHCH₂ H CH₃ H CH₃CH₂CH₂CH₂ H C₂H₅ H CH₃ H CH₃OCH₂CH₂ H CH₃ H CF₃CH₂ H CH₃ CH₃ C₂H₅ H CH₃ CH₃ CH₃CH₂CH₂ H C₆H₆CH₂ CH₃ CH₃CH₂CH₂ H C₆H₆CH₂ CH₃ CH₃CH₂CH₂CH₂ H C₆H₆CH₂ CH₃ (CH₃CH₂)(CH₃)CH H C₆H₆CH₂ CH₃ CH₃CH₂CH₂CH₂CH₂ H CH₃ H C₂H₅ CH₃ C₂H₅ H C₂H₅ CH₃


9. The method according to claim 7, wherein said cationic moiety of organic base comprises one selected from the group consisting of the following: 1-n-butyl-3-methylimidazolium (BMI), 1-octanyl-3-methylimidazolium (DMI), and 1-hexadecanyl-3-methylimidazolium (HDMI).
 10. The method according to claim 7, wherein the anionic moiety in said Lewis acid comprises one selected from the group consisting of the following: BF₄ ⁻, PF₆ ⁻, AsF₆ ⁻, SbF₆ ⁻, F(HF)_(n) ⁻, CF₃SO₃ ⁻, CF₃CF₂CF₂CF₂SO₃ ⁻, (CF₃SO₂)₂N⁻, (CF₃SO₂)₃C⁻, CF₃COO⁻, and CF₃CF₂CF₂COO⁻.
 11. The method according to claim 1, wherein the weight of said ionic liquid is about 10%-90% weight of said at least alkoxide monomer and/or aryloxide monomer.
 12. The method according to claim 1, wherein the weight of said ionic liquid is about 50%-70% weight of said at least alkoxide monomer and/or aryloxide monomer.
 13. The method according to claim 1, wherein said coating processing comprises a heating process to heat said precursor solution.
 14. The method according to claim 1, wherein the temperature of said heating process ranges from 40° C. to 70° C. to form said early stage gel.
 15. The method according to claim 1, wherein the material of said substrate comprises one selected from the group consisting of the following: silicon chip, glass, or polymer.
 16. The method according to claim 1, wherein the temperature of said gelation process ranges from 50° C. to 150° C.
 17. The method according to claim 1, wherein said gelation process comprises: a gas contacting process by having the saturated vapor of said alcohol contact with said early stage gel formed by the said precursor solution on said substrate, so as to form a stable gel layer; and a liquid contacting process by having said alcohol solution contact with said stable gel layer, so as to harden said stable gel layer without shrinkage during gelation and form said three-dimensional mesoporous layer.
 18. The method according to claim 1, wherein the number of carbon atoms in said alcohol is less than or equal to
 5. 19. The method according to claim 1, wherein the composition of said three-dimensional mesoporous layer comprises one selected from the group consisting of the following or any combination: SiO₂, TiO₂, V₂O₅, and Al₂O₃.
 20. The method according to claim 1, wherein the average pore diameter of said three-dimensional mesoporous layer ranges about 2 nm to 50 nm.
 21. The method according to claim 1, wherein the boiling point of said solvent is less than or equal to 200° C.
 22. The method according to claim 1, wherein said solvent comprises one selected from the group consisting of the following: nitrile, alcohol, ketone, and water.
 23. The method according to claim 1, wherein the temperature of said cleaning process ranges from 50° C. to 200° C.
 24. The method according to claim 1, wherein said surface modification process uses a modifier, and said modifier is an alkoxide monomer and/or aryloxide monomer with at least one specific moiety, and said specific moiety comprises one selected from the group consisting of the following: amine group, hydroxyl group, carboxyl group, and epoxy group.
 25. The method according to claim 24, wherein the method further comprises a converting process after said surface modification process, and said converting process comprises: providing a converter that comprises a first moiety and a second moiety; and bonding said specific moiety of said modifier with said first moiety of said converter to form a biochip having said second moiety on its surface.
 26. A biochip with a three-dimensional mesoporous layer, comprising a substrate covered with a three-dimensional mesoporous layer, wherein the surface of said three-dimensional mesoporous layer is modified by an alkoxide monomer and/or aryloxide monomer with at least one specific moiety and said specific moiety comprises one selected from the group consisting of the following: amine group, hydroxyl group, carboxyl group, and epoxy group.
 27. The biochip according to claim 26, wherein the specific surface area of said three-dimensional mesoporous layer is more than or equal to 100 m²/g.
 28. The biochip according to claim 26, wherein the average pore diameter of said three-dimensional mesoporous layer is less than or equal to 20 nm.
 29. The biochip according to claim 26, wherein the porosity of said three-dimensional mesoporous layer ranges about 50% to 90%. 